1. Field of the Invention
The present invention includes a method for preparing stable, long-lived gas emulsions for ultrasound contrast enhancement and other uses, and to compositions of the gas emulsions so prepared.
2. Background of the Art
Ultrasound technology provides an important and more economical alternative to imaging techniques which use ionizing radiation. While numerous conventional imaging technologies are available, e.g., magnetic resonance imaging (MRI), computerized tomography (CT), and positron emission tomography (PET), each of these techniques use extremely expensive equipment. Moreover, CT and PET utilize ionizing radiation. Unlike these techniques, ultrasound imaging equipment is relatively inexpensive. Moreover, ultrasound imaging does not use ionizing radiation.
Ultrasound imaging makes use of differences in tissue density and composition that affect the reflection of sound waves by those tissues. Images are especially sharp where there are distinct variations in tissue density or compressibility, such as at tissue interfaces. Interfaces between solid tissues, the skeletal system, and various organs and/or tumors are readily imaged with ultrasound.
Accordingly, in many imaging applications ultrasound performs suitably without use of contrast enhancement agents; however, for other applications, such as visualization of flowing blood, there have been ongoing efforts to develop such agents to provide contrast enhancement. One particularly significant application for such contrast agents is in the area of perfusion imaging. Such ultrasound contrast agents could improve imaging of flowing blood in the heart muscle, kidneys, liver, and other tissues. This, in turn, would facilitate research, diagnosis, surgery, and therapy related to the imaged tissues. A blood pool contrast agent would also allow imaging on the basis of blood content (e.g., tumors and inflamed tissues) and would aid in the visualization of the placenta and fetus by enhancing only the maternal circulation.
A variety of ultrasound contrast enhancement agents have been proposed. The most successful agents have generally consisted of dispersions of small bubbles of gas that can be injected intravenously. Most typically, the bubbles are injected into the bloodstream of a living body to be imaged. The bubbles then provide a physical object in the flowing blood that is of a different density and a much higher compressibility than the surrounding fluid tissue and blood. As a result, these bubbles can easily be imaged with ultrasound. To traverse blood vessels, the bubbles should be less than 10 μm in diameter and have been called microbubbles. Microbubbles may be formed in a liquid in a variety of different ways. Simple examples are vigorous agitation or by forcing of a gas into a liquid through a small orifice. In the absence of additional ingredients, the gas will be in direct contact with the condensed medium (i.e., naked bubbles). However, such bubbles tend to shrink rapidly due to the diffusion of the trapped gas into the surrounding liquid. In addition, “naked” microbubbles have been shown to produce adverse responses such as the activation of complement (See, for example, K. A. Shastri et al. (1991) Undersea Biomed. Res., 18, 157). Attempts to lengthen the life of microbubbles to increase their usefulness have focused on the addition of stabilizing agents which can enclose the gas bubbles, retarding the diffusion of the gas into the surrounding liquid.
Most microbubble compositions have failed to provide contrast enhancement that lasts even a few seconds, let alone minutes. This greatly limits their usefulness. Microbubbles have therefore been “constructed” in various manners in an attempt to increase their effective contrast enhancement life. Various avenues have been pursued such as the use of gelatins or albumin microspheres that are initially formed in liquid suspension, and which entrap gas during solidification. However, solid phase shells that encapsulate gases have generally proven too fragile or too permeable to the gas to have satisfactory in vivo life. Furthermore, thick shells (e.g., albumin, sugar, or other viscous materials) reduce the compressibility of the bubbles, thereby reducing their echogenicity during the short time they can exist. Solid particles or liquid emulsion droplets that evolve gas or boil when injected (as in Quay, PCT/US94/00422) pose the danger of supersaturating the blood with the gas or vapor. This will lead to a small number of large embolizing bubbles forming at the few available nucleation sites rather than the intended large number of small bubbles. In addition, bubbles created in vivo in this way will be “naked”, and consequently will have the complement activation problem described above.
The use of surfactants as stabilizing agents for gas bubble dispersions has also been explored. Surfactants are materials which tend to form an interfacial layer at the interface of a polar substance with a non-polar substance. Their “surface active” behavior arises from the existence of both a hydrophilic region (often comprising one end which is usually referred to as the “head”), which tends to associate with the polar substance, and a hydrophobic region (often comprising the other end which is usually referred to as the “tail”), which tends to associate with the non-polar substance. When established, the interfacial layer affects the characteristics of the polar/non-polar interface. When surfactants are present, the gas may be separated from the liquid by an interfacial layer which may be comprised of a wide variety of surfactant materials.
Some surfactant-containing contrast enhancement agents entrap gas bubbles in another manner, e.g., in the aqueous core of liposomes. Liposomes are more or less spherical “bags” comprised of an aqueous core bounded by one or more concentric, closed, bimolecular phospholipid layers. Phospholipids, being natural components of cell membranes, are also well known for surfactant properties. In U.S. Pat. No. 5,334,381 to Unger, liposomes containing gas bubbles are created via several different mechanisms. Also, U.S. Pat. No. 4,900,540 to Ryan et al. discloses phospholipid liposomes which contain a gas or gas precursor. Presumably, the gas bubbles trapped inside the liposomes leak out slowly, thereby increasing the efficacy of the contrast agent. It may be noted that this use of a surfactant does not involve the presence of an interfacial layer of surfactant at the gas/liquid interface. Rather, small gas bubbles are trapped in a larger volume of aqueous liquid that is itself bounded by the uni- or multi-lamellar liposomal structure.
Surfactant containing contrast agents may utilize liposomes in other ways. For example, in U.S. Pat. Nos. 5,380,519 and 5,271,928 to Schneider et al., microbubbles prepared from freeze dried liposomes are described. According to this disclosure, reconstitution in water of a dry, pulverulent formulation created by lyophilizing a liposome suspension creates a dispersion of gas bubbles in suspension with water-filled liposomes. The microbubbles so prepared are stated to be surrounded by a “rather evanescent” envelope of surfactant. Although it would generally be expected that such an evanescent surfactant layer would not have persistence, and that such microbubbles would therefore not be stable for an extended period of time, Schneider et al. theorize that the laminated surfactant in or from the neighboring water-filled liposomes stabilizes the gas present in the system in the form of microbubbles.
It is readily appreciated that a liposome dependent contrast enhancement agent requires the prior formation of liposomes, and therefore limits the main component of stabilizing surfactant to a type which is capable of forming liposomes. Moreover, liposome preparation involves sophisticated and time consuming manufacturing.
Even in the presence of stabilizing compounds or structures, the entrapped gases are under increased pressure in the bubble due to the surface tension of the surrounding surfactant, as described by the Laplace equation (ΔP=2γ/r). This increased pressure further facilitates shrinkage and disappearance of the bubble as the gas moves from a high pressure area (in the bubble) to a lower pressure environment (in either the surrounding liquid which is not saturated with gas at this elevated pressure, or into a larger diameter, lower pressure bubble).
One proposal for dealing with such problems is outlined in Quay, PCT/US92/07250. Quay forms bubbles using gases selected on the basis of being a gas at body temperature (37° C.) and having reduced water solubility, higher density, and reduced gas diffusivity in solution in comparison to air. Although reduced water solubility and diffusivity can affect the rate at which the gas leaves the bubble, numerous problems remain with the Quay bubbles. Forming bubbles of sufficiently small diameter (e.g., 3–5 μm) requires high energy input. This is a disadvantage in that sophisticated bubble preparation systems must be provided at the site of use. Moreover, The Quay gas selection criteria are incorrect in that they fail to consider certain major causes of bubble shrinkage, namely, the effects of bubble surface tension, surfactants and gas osmotic effects, and these errors result in the inclusion of certain unsuitable gases and the exclusion of certain optimally suitable gases.
Accordingly, a need exists in the art for compositions, and a method to prepare such compositions, that provide, or utilize, a longer life contrast enhancement agent that is biocompatible, easily prepared, and provides superior contrast enhancement in ultrasound imaging.